Immunoassay Kit (Colorimetric)

RayBio cgmp Direct Immunoassay Kit (Colorimetric) User Manual Version 1.0 May 25, 2014 RayBio cgmp Direct Immunoassay Kit (Colorimetric) Protocol (Cat#: 68AT-cGMP-S100) RayBiotech, Inc. We Provide You With Excellent Support And Service

Tel:(Toll Free)1-888-494-8555 or 770-729-2992; Fax:770-206-2393; Web: www.raybiotech.com Email: info@raybiotech.com RayBiotech, Inc. RayBio cgmp Direct Immunoassay Kit (Colorimetric) TABLE OF CONTENTS I. Introduction. 1 II. Kit Contents. 2 III. Assay Protocol... 2 I. INTRODUCTION Adenosine and guanosine 3,5 -cyclic monophosphate (camp and cgmp) are important second messengers involved in many physiological processes. RayBiotech s cgmp Direct Immunoassay Kit provides a direct competitive immunoassay for sensitive and quantitative determination of cgmp level in biological samples. The kit utilizes the recombinant Protein G coated plate to anchor cgmp polyclonal antibody. cgmp-hrp conjugates directly competes with cgmp from samples for binding to the cgmp specific antibody on the plate. After incubation and washing, the amount of cgmp-hrp bound to plate can easily be determined by reading OD450 nm. The intensity of OD450 nm is inversely proportional to the concentration of cgmp in samples. The kit provides a new acetylation procedure that improves detection signal significantly. The kit can detect 0.04-10 pmol/well (0.008-2 μm) cgmp samples. RayBio cgmp Direct Immunoassay Kit 1

II. KIT CONTENTS Store kit at -20 C Component 68AT-cGMP-S100 Color Code Storage Part 100 assays Cap Color Temperature Number 10X cgmp Assay Buffer Standard cgmp (10 nmol) Neutralizing Buffer Acetylating Reagent A Acetylating Reagent B Rabbit Anti-cGMP pab/bsa cgmp-hrp/bsa HRP Developer Protein G Coated Plate 25 ml 1 vial 7.5 ml 0.75 ml 1.5 ml 1 vial 1 vial 10 ml 1 each WM Yellow NM Violet Black Red Green Amber ------ -20 C -20 C -20 C -20 C Item A Item B Item C Item D Item E Item F Item G Item H Item I III. Assay Protocol A. Reagent Preparations: Dilute the 10X cgmp Assay Buffer to 1X Assay Buffer with MilliQ water. Store at 4 C. Reconstitute the Standard cgmp (pellet may not be visible) in 1 ml of 0.1M HCl (not provided), vortex for 10 seconds for a 10 pmol/μl cgmp stock standard solution. Reconstitute rabbit anti-cgmp pab and cgmp-hrp each with 1.1 ml of the 1X Assay Buffer as stock solutions. Unused well strips can be kept at 20 C with the desiccants, stable for up to 1 month. The kit should be stored at 20 C. After reconstitution, some components may be stored at 4 C as instructed above, stable for up to 1-2 months. *NOTE- Acetylating Reagent B is very volatile and hence the vial has to be tightly capped and stored only at. RayBio cgmp Direct Immunoassay Kit 2

B. General Consideration: cgmp samples in 0.1 M HCl (final concentration) is stable and can be used directly in the assay. Make dilutions of your sample with 0.1 M HCl to the range of 0.04-10 pmol/well (0.008-2 μm). Plasma, serum, whole blood, and tissue homogenates often contain phosphodiesterases and large amount of immunoglobulins (Igs) which may interfere with the assay. However, preparing samples in 0.1 M HCl can generally inactivate phosphodiesterases and lower the concentration of Igs, making the samples suitable for the assay. Both phosphodiesterases and Igs can also be removed by 5% TCA precipitation or by using 10 Kd molecular weight cut off microcentrifuge filters. Organic solvents in samples may interfere with the assay, which may need to be removed prior to the assay. To determine whether interference is presence in your sample, you may make two different dilutions. If the two different dilutions of sample show good correlation in the final calculated cgmp concentrations, purification is not required. If you do not see good correlation of the different dilutions, deproteinize the sample by using TCA or 10 Kd molecular cut off microcentrifuge filters. Some organic solvents may interfere with the assay and may need to be removed prior to the assay. C. Sample Preparation: Urine, Plasma and Culture Medium Samples: Urine, plasma, and culture media may be tested directly after adding 1/10 volume of 1M HCl, and remove precipitates if they occur. Culture Cells: For suspension cells; collect by centrifugation. Add 1mL of 0.1M HCl for every 35cm 2 of surface area (e.g., 10cm plate at 70% confluency is ~110cm 2, so use ~3.1mL). For Adherent Cells: add the HCl directly, scrape cells off the surface. Dissociate sample by pipetting up and down until suspension is homogenous. Transfer to a centrifuge tube and centrifuge at top speed for 10 min. The supernatant can be assayed directly. Protein concentration of >1 mg/ml is recommended for reproducible results. Tissue Samples: Cyclic nucleotides may be metabolized quickly in tissue, so it is important to rapidly freeze tissues after collection (e.g., using liquid nitrogen). Weigh the frozen tissue and add 5-10 volume of 0.1M HCl. Homogenize the RayBio cgmp Direct Immunoassay Kit 3

sample on ice using a Polytron-type homogenizer. Spin at top speed for 5 min and collect the supernatant. The supernatant may be assayed directly. D. cgmp Assay: The procedure described here includes an acetylation step which makes the cgmp assay much more sensitive and avoid the interferences of many components in samples. However, for routine assay of the well known samples, non-acetylation procedure may also be used, just skip the acetylation steps (Step 7 and 8). Prepare cgmp Standard Curve and Samples: 1. Add 200 μl of the 10 pmol/μl standard cgmp stock into 800 μl of 0.1M HCl to generate 2 pmol/μl cgmp working solution. The diluted cgmp should be used within 1 hour. 2. Label 11 microcentrifuge tubes, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.039, 0, 0_B pmol/50 μl. (Note: these concentrations represent what will finally be in the wells after the dilutions mentioned below). 3. Add 200 μl of the 2 pmol/μl cgmp into the tube labeled 10 pmol (enough for 20 tests), add 100 μl 0.1M HCl into the rest of tubes. 4. Transfer 100 μl from the 10 pmol tube into the labeled 5 pmol tube, mix. Continue the serial dilution by transferring 100 μl from the 5 pmol tube to 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078, 0.039 pmol tubes. Discard 100 μl from the 0.039 pmol tube. The diluted cgmp should be used within 1 hour. 5. Label new tubes for test samples, add 100 μl each test sample per tube. We suggest using different dilutions for each sample (dilute with 0.1M HCl). 6. Add 50 μl of Neutralizing Buffer to each tube to neutralize the HCl in the samples and standards. 7. Prepare Acetylating Reagent Mix (Note: 5 μl is needed for each assay): Mix 1 volume of Acetylating Reagent A (Violet cap) with two volumes of Acetylating Reagent B (Black cap) in a microtube. Prepare just enough for the experiment. Use within 1 hour. 8. Add 5 μl of the Acetylating Reagent Mix directly into each test solution (both standard and sample), IMMEDIATELY vortex 2-3 seconds following each addition without delay, one tube at a time and incubate at room temperature for 10 min. 9. Add 845 μl 1X Assay Buffer into each tube, mix well. Use for below quantification. Note: The acetylation step improves the assay sensitivity significantly and avoid the interferences of many components in unpurified samples. (If cgmp in your RayBio cgmp Direct Immunoassay Kit 4

samples are very low, the acetylation reagents can be dried after step 8, without dilution step 9 to minimize the volume. Then reconstituted in a 50-100 μl volume of Assay Buffer). Quantification cgmp: 1. Add 50 μl of the acetylated Standard cgmp and test samples from Step 9 above to each well of the Protein G coated 96-well plate. We suggest duplicate assays for each sample and standard. 2. Add 10 μl of the reconstituted cgmp antibody per well to the standard cgmp and sample wells except the well with 0_B pmol cgmp. (Note: Do not add cgmp antibody into the well with 0_B pmol cgmp, instead add 10 μl of 1X Assay Buffer for background reading). Incubate for 1 hour at room temperature with gentle agitation. Note: Using a repeating pipette is recommended for minimizing pipetting errors. 3. Add 10 μl of cgmp-hrp to each well and incubate for 1 hr at room temperature with gentle agitation. 4. Wash 5 times with 200 μl 1X Assay Buffer each time. Completely empty the wells by tapping the plate on a fresh paper towel after each wash step. 5. Add 100 μl of HRP developer and develop for 1 hour at room temperature with agitation. 6. Stop the reaction by adding 100 μl of 1M HCl (not provided) to each well (sample color should change from blue to yellow). 7. Read sample at OD 450 nm. 8. Subtract OD450 nm background reading (the well with 0_B pmol cgmp) from all samples and standards. Plot standard curve to observe the linear portion, then replot only the linear portion and in Excel add a trendline, then use the trend line linear formula (y=mx+b). Calculate amount of cgmp in samples after correcting the for dilution factors. 9. Calculations: cgmp Concentration = Sa/Sv (pmol/µl or nmol/ml or µm) Where: Sa is cgmp amount (pmol) from standard curve. Sv is sample volume (µl) added into the assay wells after dilution factor correction. RayBio cgmp Direct Immunoassay Kit 5

cgmp Standard Curve: The assay was performed following the kit protocol. RayBio cgmp Direct Immunoassay Kit 6

GENERAL TROUBLESHOOTING GUIDE: Problems Cause Solution Assay not working Use of ice-cold assay buffer Assay buffer must be at room temperature Samples with erratic readings Lower/ Higher readings in Samples and Standards Readings do not follow a linear pattern for Standard curve Omission of a step in the protocol Plate read at incorrect wavelength Use of a different 96-well plate Use of an incompatible sample type Samples prepared in a different buffer Samples were not deproteinated Cell/ tissue samples were not completely homogenized Samples used after multiple free-thaw cycles Presence of interfering substance in the sample Use of old or inappropriately stored samples Improperly thawed components Use of expired kit or improperly stored reagents Allowing the reagents to sit for extended times on ice Incorrect incubation times or temperatures Incorrect volumes used or protocol properly followed Use of partially thawed components Pipetting errors in the standard Pipetting errors in the reaction mix Air bubbles formed in well Standard stock is at an incorrect concentration Calculation errors Substituting reagents from older kits/ lots Refer and follow the data sheet precisely Check the wavelength in the data sheet and the filter settings of the instrument Fluorescence: Black plates (clear bottoms) ; Luminescence: White plates ; Colorimeters: Clear plates Refer data sheet for details about incompatible samples (Samples should initially be at 1 mg/ml protein) Use the assay buffer provided in the kit or refer data sheet for instructions Use the 10 kda spin cut-off filter or TCA precipitation as indicated or use 0.1 M HCl to inactivate phosphodiesterases Use Dounce homogenizer (increase the number of strokes); observe for lysis under microscope Aliquot and freeze samples if needed to use multiple times Troubleshoot if needed, deproteinize samples Use fresh samples or store at correct temperatures till use Thaw all components completely and mix gently before use Always check the expiry date and store the components appropriately Always thaw and prepare fresh reaction mix before use Refer datasheet & verify correct incubation times and temperatures Samples/standards aceylated and treated one at a time through this treatment. Use calibrated pipettes and aliquot correctly Thaw and resuspend all components before preparing the reaction mix Avoid pipetting small volumes Prepare a master reaction mix whenever possible Pipette gently against the wall of the tubes Always refer the dilutions in the data sheet Recheck calculations after referring the data sheet; Use only the linear portion of curve. Use fresh components from the same kit Unanticipated results Measured at incorrect wavelength Check the equipment and the filter setting Samples contain interfering substances Use of incompatible sample type Sample readings above/below the linear range Troubleshoot if it interferes with the kit Refer data sheet to check if sample is compatible with the kit or optimization is needed Concentrate/ Dilute sample so as to be in the linear range Note: The most probable list of causes is under each problem section. Causes/ Solutions may overlap with other problems. RayBio cgmp Direct Immunoassay Kit 7

This product is for research use only. 2004 RayBiotech, Inc. RayBio cgmp Direct Immunoassay Kit 8